CN101291951A - Method for manufacturing high purified factor IX - Google Patents
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- CN101291951A CN101291951A CNA2006800390241A CN200680039024A CN101291951A CN 101291951 A CN101291951 A CN 101291951A CN A2006800390241 A CNA2006800390241 A CN A2006800390241A CN 200680039024 A CN200680039024 A CN 200680039024A CN 101291951 A CN101291951 A CN 101291951A
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Abstract
The present invention discloses a preparation method of a highly purified human blood coagulation factor IX. The human blood coagulation factor IX is prepared by performing anion exchange chromatography, cation exchange chromatography, heparin affinity chromatography to the material containing human blood coagulation factor IX (taken from human blood serum or recombined cell culture medium), which includes the period of passivating virus through S/D treatment (Solvent/Detergent treatment) or removing virus through nanofiltration. The highly purified safe coagulation factor IX preparation having a specific activity of above 150 IU/mg, with substantially undoped proteins can be prepared by the preparation method of the invention.
Description
Technical field
The present invention relates to a kind of preparation method of highly purified people IX thrombin, be particularly related to a kind ofly, can make the almost high purity IX thrombin of non-impurity-doped protein, the safety of specific activity (specific activity) more than 150IU/mg by the material (taking from the cell culture medium of human plasma or reorganization) that comprises the IX thrombin being carried out the stage that ion exchange chromatography and affinity chromatography make viral passivation or remove virus.
Background technology
People IX thrombin is a glycoprotein indispensable in the coagulation process, because inherited genetic factors or pathological factor and do not have under IX thrombin or the insufficient situation of IX thrombin in the blood, can make the coagulation process in the blood imperfect, thus inducing B type hemophilia.
Haemophilia B mainly takes place by heredity, and 30,000～50,1 boy baby has haemophilia B among 000 boy baby, belongs to rare heredopathia.For treating the method that these haemophilia Bs patient has adopted purifying and the IX factor offers patient's body in the blood of human body after concentrating.
Required vitamin b6 usp K when the synthetic IX factor of liver has multiple and other vitamin b6 usps K-dependency glycoprotein common feature.The vitamin b6 usp K-dependency glycoprotein that participates in coagulation process has the II factor, the VII factor, the X factor etc.The IX factor is the glycoprotein that contains the monomer structure (monomer) of 17.5% the sugar (4.7% hexose, 6.8%N-acetylamino hexose, 6% sialic acid) of having an appointment, and belongs to a kind of of serine protease (serine protease).Molecular weight is determined that by the carbohydrate amount in the IX factor its size is about 55,000～75,000Da.Be about 47 according to the independent protein molecular weight that aminoacid sequence calculated, 054Da.There is zone near the N-terminal in molecular structure, when activation, needs phosphatide (phospholipid) and calcium ion with 12 gamma-carboxyl glutamates (carboxyglutamic acid).Owing to contain with per 1 molecule IX factor and to contain the sialic acid (sialic acid) that belongs to acidic oligomer sugar (acidic oligosaccharide) about 10 molecules, therefore have very low iso-electric point (isoelectric point, pI).According to the kind of the hydrocarbon polymer that exists in the IX factor and amount show pl be 4.0～4.6 diversity (Journal ofchromatography A, 844,1999,119-128).
By between amino acid Arg (the 145)～Ala (146) of the activatory XIa factor (Factor XIa) the hydrolysis IX factor and between Arg (180)～Val (181), the active peptide (activation peptide) that is made of 35 amino acid breaks away from and activates, and is called the active form (active form) of the IX factor for the IX factor that will break away from active peptide with natural IX factor difference.And also can be by comprising the activatory VIIa factor (Factor VIIa), tissue factor (Factor III) and calcium ion (Ca
2+) phospholipid complex activate (as shown in Figure 1).The concentration that is stored in the IX factor in the human plasma is 0.1 μ mol (5 μ g/ml), thereby has lower concentration.Thus, for treatment haemophilia B patient's treatment need provide a large amount of whole blood or blood plasma, can excessively provide other protein of the Fibrinogen (fibrinogen) that is stored in the blood plasma etc. simultaneously.
For reducing these side effects, nineteen fifty is developed the spissated IX factor first for the end.This method is that post precipitation is removed other protein by washing by the IX factor in the blood plasma being adsorbed in barium sulfate (barium sulfate), after this isolates the IX factor that is adsorbed in barium sulfate.Finally, the tricalcium phosphate (tricalcium phosphate) with the nontoxicity salt has replaced barium sulfate.This by in the absorption and the enrichment process of the sedimentary IX factor reconcentration IX factor time, the II factor, the VII factor, the X factor in can concentrated simultaneously other vitamin b6 usps K-dependency glycoprotein.This enriched material is called IX factor mixture (Factor IXcomplex).Since nineteen sixty for, spissated by the way IX factor mixture generally is used for the treatment of haemophilia B.
General by existing albumin or the sphaeroprotein of isolating in the cold ethanol fractionation operation in the blood plasma of stating, but IX factor mixture can not be separated by this cold ethanol separation circuit, thus be not suitable for the preparation of the IX factor.For solving above-mentioned shortcoming, develop the column chromatography operation of utilizing anion-exchange chromatography.Promptly, by cold heavy (Cryoprecipitation) remove have the cryoprecipitate (cryoprecipitate) of the abundant VIII factor after, be adsorbed in the spissated method of anionite-exchange resin purifying with removing IX factor cryoprecipitate, in the blood plasma.But cause concentrating of the II factor, the VII factor, the X factor in the method too.This IX factor mixture that utilizes anionite-exchange resin to produce also is usually used in the treatment of haemophilia B till now.
But, after being provided, IX factor mixture has phlebothrombosis disease (venous thrombosis) and disseminated inravascular coagulation (disseminated intravascularcoagulation, symptom (Thrombosis ﹠amp such as DIC) according to report; Haemostasis, vol 73, no.4,584-591, vol 79, no.4,778-783).By inference, this side effect is because (the hypercoagulable state) that unnecessary thrombogen composition (thrombogenic components) is caused in other too much solidifying proteins that provide simultaneously with the IX factor or the enriched material produces.Too much or provide for a long time normal II factor amount is increased, the time that it is disappeared in blood plasma is elongated.
For reducing the side effect of IX factor mixture, since the nineteen ninety production high purity IX factor, this high purity IX factor has been removed the II factor, the VII factor, the X factor.The high purity IX factor, be by carrying out anion-exchange chromatography, spissated IX factor mixture is carried out the grand antibody gel filtration chromatography (monoclonal antibody gel column chromatography) of heparin gel method (Heparin gel) or monomer and removed the II factor, the VII factor, the X factor, thereby replaced IX factor mixture with contact.And Genetic Institute developed the genetic recombination IX factor of utilizing CHO cell in 1997, and with Benefix
TMTitle is sold.
The disadvantage that derives from the thrombin of blood is the danger that has virus infection, and this virus is meant HIV, HBV, HCV that might be in blood etc.Virus safe for the thrombin of guaranteeing to derive from blood must comprise the operation of removing virus at purifying and in concentrating.Generally handle and make viral passivation, disclose the operation of in passivation, carrying out affinity chromatography for further effectively removing virus by S/D.And, also utilize nanofiltration (nanofilter, more efficiently nanofiltration process 20nm), but when utilizing this nanofiltration, need to carry out the spissated operation of high purity to thrombin in advance, do not carry out the thrombin of prepared in high purity filter stoppage and can not using when carrying out nanofiltration to prevent to occur in utilization.
Though can need use zooblast owing to produce antibody, but can not get rid of the virus infection that derives from zooblast by grand antibody (monoclonal antibody) the selective separation IX factor of monomer.And, can not get rid of in carrying out the chromatography operation the caused side effect of immunoglobulin (Ig) (IgG) with the synantibody elution.And, can not overcome the virus problems when from the reconstitution cell substratum, separating the IX factor fully.Thereby need develop and a kind ofly can remove virus fully, and purity is greater than the preparation method of the people IX thrombin of 99% safety.
Summary of the invention
Be the deficiency that addresses the above problem, the present invention phlebothrombosis disease (venousthrombosis) due to the hypercoagulative state (hypercoagulable state) that is caused by unnecessary thrombosis composition in unnecessary clottable protein warmed that is provided in hemophilia B patient's the treatment or the enriched material or disseminated intravascular coagulation is provided (disseminated intravascularcoagulation, DIC) preparation method of the highly purified people IX thrombin of the side effect of Denging is a purpose to provide a kind of.
Below the preparation method of highly purified people IX thrombin of the present invention is described.
Fig. 2 is a people IX thrombin preparation method's of the present invention schema.
The sample that is used for solution such as human plasma of the present invention, thrombin is not limited to the material of specified shape.The preparation method's of people IX thrombin of the present invention raw material is preferably the human plasma of removing cryoprecipitate (cryo), also comprise the blood plasma part that comprises the IX factor, reorganization the IX factor cell culture medium or comprise the material etc. of the IX factor.
In one embodiment of this invention, carrying out carrying out after anion-exchange chromatography (anion exchangechromatography) comes elution to comprise the solution of the IX factor carrying out concentrating after anion-exchange chromatography comes elution again after S/D (Solvent/Detergent treatment) handles passivation virus and filtration prepares in the method for people IX thrombin, further comprise, to solution by the anion-exchange chromatography elution, carry out the heparin affinity chromatography and carry out elution, then collect non-adsorption liquid, carry out the operation that nanofiltration (nanofiltration) is removed virus by carrying out cation-exchange chromatography.
Be specially, in the present invention for removing with other residual doping protein in the plasma protein solution of above-mentioned anion-exchange chromatography preparation, the heparin (heparin) that carries out thrombin is had affinity is the heparin affinity chromatography of ligand, collects the non-adsorption liquid that is not adsorbed in cationic exchange coloum and removes other impurity.
In the above-mentioned heparin affinity chromatography because ligand heparin (heparin), its carrier (resin) can utilize various kinds of resin such as sepharose, agarose, polyacrylamide, but is not limited to this.Override uses widely used heparin sepharose 6FF post (heparinsepharose 6FF).In heparin affinity chromatography of the present invention, the ionic strength (ionic strength) of employed level pad and the ionic strength that comprises the solution of the IX factor are below the 20mS/cm before being suitable for the solution comprise the IX factor, pH is 5.0～9.0, the ionic strength of the lavation buffer solution when washing is adsorbed in other protein of resin is 10～20mS/cm, and the ionic strength of the elution damping fluid when elution then comprises the solution of the IX factor is 20～50mS/cm.
And, the post functional group (functional group) of wherein said cation-exchange chromatography (cation exchangechromatography) be selected from the weak cation (weakcation) carboxymethyl (carboxymethyl-, CM-), carboxyl (carboxy-, C-), the sulfo group (sulfo-in the strong cation (strong cation), S-), sulfonic acid methyl (sulfomethyl, SM-), sulfoethyl (sulfoethyl-, SE-), sulfopropyl (sulfopropyl-, SP-), phospho (phospho-, and be not limited to this P-) etc..Post resin (resine) is selected from sepharose (Sepharose), dextran (Sephadex), agarose (agarose), Sephacel (Sephacel), polystyrene (Polystyrene), polyethylene (Polyacrylate), Mierocrystalline cellulose (Cellulose), Toyoperl etc., and is not limited to this.Above-mentioned cation-exchange chromatography among the present invention preferably carries out under pH 3.0～6.0 conditions, and is not adsorbed in the non-adsorption liquid that post spills and prepares by collecting.Level pad in the cation-exchange chromatography can use in many ways according to condition, and the ionic concn that the ionic concn (ionic strength) of employed level pad and use comprise the solution of the IX factor before being suitable for the solution comprise the IX factor is preferably 10～50mS/cm.
The present invention relates to a kind of preparation method of highly purified people IX thrombin, be particularly related to a kind ofly, can make the almost high purity IX NiaState of non-impurity-doped protein, the safety of specific activity (specific activity) more than 150IU/mg by the material (taking from the cell culture medium of human plasma or reorganization) that comprises the IX thrombin being carried out the stage that ion exchange chromatography and heparin affinity chromatography make viral passivation or remove virus.
Description of drawings
Fig. 1 is the synoptic diagram of expression intrinsic coagulation approach and exogenous cruor pathway.
Fig. 2 is a highly purified people IX thrombin preparation method's of the present invention synoptic diagram.
Fig. 3 be illustrated in non-reduced (A) state and, the SDS-PAGE result's that carries out under reduction (B) state, to XI NiaState synoptic diagram by prepared high purity XI NiaState of the present invention and market sale.
M: standard label (myosin; 198, nougat; 115, BSA; 93, ovalbumin; 49.8, carbonic anhydrase; 35.8, Trypsin inhibitor SBTI; 29.2, lysozyme; 21.3, Trypsin inhibitor,Trasylol; 6.4); Lane 1: according to the high purity XI thrombin of the method for table 2; Lane 2: according to the high purity XI thrombin of the method for table 3; Lane 3: according to the high purity XI thrombin of the method for table 4; Lane 4:Facnyne (The Green Cross Corporation); Lane 5:Mononine (ZLB Behring company); Lane 6:Octanyne (Octapharma company); Lane 7:Berinin HS 600 (ZLB Behring company); Lane 8:Immunine STIM plus 600 (Baxter company)
Fig. 4 is that expression is according to the activity by pH (activity) the safety results synoptic diagram of completing methyl-sepharose FF.
Embodiment
Embodiment 1: the purifying (conventional processes) that utilizes anion-exchange chromatography
With the blood plasma of removal cryoprecipitate or the cell culture medium of the IX factor of recombinating is raw material, this raw material is mixed with diethylin-ethyl-glucose gel A-50 (DEAE-Sephadex A-50), after damping fluid prewashing (prewash), under 4 ℃ of temperature condition, stirred 2 hours.Then the glue that is adsorbed with the IX factor is filtered and the isolating mode in the center of circle is separated.After prewashing (washing), the IX factor is by the damping fluid elution of high salt concentration.The pH value of above-mentioned washings suitably is adjusted to about 7.5, then dialyses, concentrate and under-70 ℃ of conditions, preserve.And, separately concentrated solution is taken a sample (aliquot), the Blood Preparations by KFDA (Korea Food and Drug Administration) regulation and the standard of recombination preparation and determination of experimental method concentration.The part that comprises the IX factor is carried out S/D to be handled and finishes the viral purification operation.With finishing solution absorbs after the purification procedures after 2 purifying are with DEAE-toyopearl 650M post, collect eluant according to operation.
Embodiment 2: the purifying that utilizes heparin sepharose (Heparin sepharose) 6FF column chromatography
To be adjusted to below the ionic strength 20mS/cm by the IX proconvertin solution 10ml of the foregoing description 1 purified preparation, behind the pH7.0, at heparin sepharose 6FF post (column condition: diameter 1cm, height 18cm, flow velocity (flow rate) 1.0ml/min, normal temperature) flow into level pad (20mM Trisodium Citrate, pH 7.0) keep balance, and sample dress sample (l0ading sample) is flow through and adsorb, by carrying out once washing with the solution identical with level pad, after the sodium-chlor of one 0.1M carries out secondary washing, with the sodium-chlor elution of 0.25M.Above-mentioned eluant is carried out having carried out SDS-PAGE behind the determination of protein concentration, and (BSA standard (2mg/ml, PIERCE)) utilizes the Coagulation timer KC10 of Amelung company to measure concentration.Specific activity to the heparin sepharose 6FF post eluant of 2 the anion column washingss of embodiment 1 and embodiment 2 compares, find that the specific activity in the heparin sepharose 6FF post operation is 80-90IU/mg, be more than 14 times of eluant concentration of embodiment 1.
Embodiment 3: the purifying that utilizes cation-exchange chromatography (CM-sepharose FF)
To be adjusted to below the ionic strength 20mS/cm by the foregoing description 2 purified IX proconvertin 10ml, behind the pH4.0, be applicable to level pad (Citrate trianion accounts for 20mM, pH 4.0 in 0.30～0.4M sodium-chlor) keep equilibrated CM-sepharose FF post (column condition: diameter 1cm, the height 5cm, flow velocity (flow rate) 1.0ml/min) after collected non-adsorption liquid.Above-mentioned non-adsorption liquid is carried out having carried out SDS-PAGE behind the determination of protein concentration, and the discovery specific activity is minimum to be more than the 150IU/mg.
Fig. 3 is the synoptic diagram that is expressed as the SDS-PAGE result of the purity of confirming other products by prepared XI NiaState of the present invention and market sale.
As shown in Figure 3, the IX NiaState of purified preparation has the doping protein of comparing with other products still less according to the present invention, thereby IX thrombin of the present invention as can be known has better purity.
Be to confirm the feasibility of mass production high purity of the present invention IX NiaState, carried out repeatedly enlarging experiment, and with the specific activity in each stage, purifying doubly, output etc. is listed in the table below in 1～6.
[table 1]
| Volume (ml) | Active (IU/ml) | Protein (mg/ml) | Gross activity (IU) | Gross protein (mg) | Specific activity (IU/mg) | Purifying doubly | Output (%) | |
| 1 resin anion(R.A) eluant | 9000 | 74.1 | 55.80 | 666900 | 502200 | 1.33 | 1 | 100 |
| The S/D treatment solution | 32700 | 18.6 | 13.05 | 606585 | 426702 | 1.43 | 1.1 | 91.0 |
| 2 resin anion(R.A) eluants | 8400 | 32.5 | 5.65 | 272580 | 47485 | 5.75 | 4.3 | 40.9 |
| Affinity resin wash extract | 24600 | 9.8 | 0.11 | 241818 | 2681 | 89.09 | 67.0 | 36.3 |
| The non-adsorption liquid concentrated solution of resin cation (R.C.) | 1500 | 127.8 | 0.76 | 191700 | 1143 | 168.15 | 126.4 | 28.7 |
| Nanofiltration solution | 3000 | 64.4 | 0.39 | 193200 | 1164 | 165.64 | 124.5 | 29.0 |
| Dialysis concentrated solution (thick concentrated solution) | 1500 | 126.6 | 0.67 | 189900 | 1004 | 188.96 | 142.1 | 28.5 |
| Final concentrated solution | 1500 | 117.3 | 0.61 | 175950 | 920 | 192.30 | 144.6 | 26.4 |
[table 2]
| Volume (ml) | Active (IU/ml) | Protein (mg/ml) | Gross activity (IU) | Gross protein (mg) | Specific activity (IU/mg) | Purifying doubly | Output (%) | |
| 1 resin anion(R.A) eluant | 9000 | 68.3 | 44.60 | 614700 | 401400 | 1.53 | 1 | 100 |
| The S/D treatment solution | 26100 | 5.2 | 16.01 | 396720 | 418461 | 0.95 | 0.6 | 64.5 |
| 2 resin anion(R.A) eluants | - | - | - | - | - | - | - | - |
| Affinity resin wash extract | 37020 | 9.3 | 0.14 | 344286 | 5183 | 66.43 | 43.4 | 56.0 |
| The non-adsorption liquid concentrated solution of resin cation (R.C.) | 2000 | 156.4 | 0.87 | 312800 | 1740 | 179.77 | 117.5 | 50.9 |
| Nanofiltration solution | 2500 | 17.2 | 0.65 | 293000 | 1625 | 180.30 | 117.8 | 47.7 |
| Dialysis concentrated solution (thick concentrated solution) | - | - | - | - | - | - | - | - |
| Final concentrated solution | 2800 | 97.4 | 0.56 | 272720 | 1568 | 173.93 | 113.7 | 44.4 |
[table 3]
| Volume | Active | Protein | Gross activity | Gross protein | Specific activity | Purifying doubly | Output |
| (ml) | (IU/ml) | (mg/ml) | (IU) | (mg) | (IU/mg) | (％) | ||
| 1 resin anion(R.A) eluant | 9000 | 65.6 | 54.03 | 590400 | 486270 | 1.21 | 1 | 100 |
| The S/D treatment solution | 31500 | 18.1 | 14.09 | 570150 | 443835 | 1.28 | 1.1 | 96.6 |
| 2 resin anion(R.A) eluants | 10050 | 43.4 | 9.67 | 436170 | 97184 | 4.49 | 3.7 | 73.9 |
| Affinity resin wash extract | 37000 | 10.6 | 0.14 | 392200 | 5180 | 75.71 | 62.6 | 66.4 |
| The non-adsorption liquid concentrated solution of resin cation (R.C.) | 2500 | 112.8 | 0.59 | 282000 | 1475 | 191.19 | 158.0 | 47.8 |
| Nanofiltration solution | 5000 | 59.8 | 0.31 | 299000 | 1550 | 192.90 | 159.4 | 50.6 |
| Dialysis concentrated solution (thick concentrated solution) | 2500 | 113.6 | 0.56 | 284000 | 1400 | 202.86 | 167.7 | 48.1 |
| Final concentrated solution | 2500 | 107.7 | 0.56 | 269250 | 1400 | 192.32 | 158.9 | 45.6 |
[table 4]
| Volume (ml) | Active (IU/ml) | Protein (mg/ml) | Gross activity (IU) | Gross protein (mg) | Specific activity (IU/mg) | Purifying doubly | Output (%) | |
| 1 resin anion(R.A) eluant | 18000 | 69.4 | 58.70 | 1249200 | 1056600 | 1.18 | 1 | 100 |
| The S/D treatment solution | 65400 | 9.1 | 13.35 | 595140 | 873090 | 0.68 | 0.6 | 47.6 |
| 2 resin anion(R.A) eluants | 16300 | 49.3 | 12.33 | 803590 | 200979 | 4.00 | 3.4 | 64.3 |
| Affinity resin Xian extract | 27500 | 24.6 | 0.28 | 676500 | 7700 | 87.86 | 74.5 | 54.2 |
| The non-adsorption liquid concentrated solution of resin cation (R.C.) | 5000 | 133.7 | 0.74 | 668500 | 3700 | 180.68 | 153.1 | 53.5 |
| Nanofiltration solution | 5075 | 111.8 | 0.69 | 567385 | 3502 | 162.02 | 137.3 | 45.4 |
| Dialysis concentrated solution (thick concentrated solution) | 3940 | 145.0 | 0.79 | 571300 | 3113 | 183.52 | 155.5 | 45.7 |
| Final concentrated solution | 3900 | 144.5 | 0.74 | 563550 | 2886 | 195.27 | 165.5 | 45.1 |
[table 5]
| Volume (ml) | Active (IU/ml) | Protein (mg/ml) | Gross activity (IU) | Gross protein (mg) | Specific activity (IU/mg) | Purifying doubly | Output (%) | |
| 1 resin anion(R.A) eluant | 17500 | 73.7 | 50.90 | 1289750 | 890750 | 1.45 | 1 | 100 |
| The S/D treatment solution | 59300 | 12.5 | 15.11 | 741250 | 896023 | 0.83 | 0.6 | 57.5 |
| 2 resin anion(R.A) eluants | 17000 | 41.2 | 9.76 | 700400 | 165920 | 4.22 | 2.9 | 54.3 |
| Affinity resin Xian extract | 34000 | 16.5 | 0.25 | 561000 | 8500 | 66.00 | 45.5 | 43.5 |
| The non-adsorption liquid concentrated solution of resin cation (R.C.) | 5000 | 59.8 | 0.44 | 299000 | 2200 | 135.90 | 93.7 | 23.2 |
| Nanofiltration solution | 5000 | 61.2 | 0.41 | 306000 | 2050 | 149.27 | 102.9 | 23.7 |
| Dialysis concentrated solution (thick concentrated solution) | 3000 | 105.1 | 0.63 | 315300 | 1890 | 166.83 | 115.1 | 24.4 |
| Final concentrated solution | 3000 | 106.9 | 0.63 | 320700 | 1890 | 169.68 | 117.0 | 24.9 |
[table 6]
| Volume (ml) | Active (IU/ml) | Protein (mg/ml) | Gross activity (IU) | Gross protein (mg) | Specific activity (IU/mg) | Purifying doubly | Output (%) | |
| 1 resin anion(R.A) eluant | 17200 | 71.9 | 53.30 | 1236680 | 916760 | 1.35 | 1 | 100 |
| The S/D treatment solution | 60820 | 13.9 | 15.02 | 845398 | 913516 | 0.93 | 0.7 | 68.4 |
| 2 resin anion(R.A) eluants | 17000 | 49.3 | 11.01 | 838100 | 187170 | 4.48 | 3.3 | 67.8 |
| Affinity resin wash extract | 30400 | 18.7 | 0.28 | 568480 | 8512 | 66.79 | 49.5 | 46.0 |
| The non-adsorption liquid concentrated solution of resin cation (R.C.) | 4500 | 104.3 | 0.62 | 469350 | 2790 | 168.22 | 124.6 | 38.0 |
| Nanofiltration solution | 4300 | 100.3 | 0.61 | 431290 | 2623 | 164.43 | 121.8 | 34.9 |
| Dialysis concentrated solution (thick concentrated solution) | 4000 | 106.5 | 0.61 | 426000 | 2440 | 174.59 | 129.3 | 34.4 |
| Final concentrated solution | 4000 | 97.4 | 0.60 | 389600 | 2400 | 162.33 | 120.2 | 31.5 |
Shown in table 1～6, repeated repeatedly the present invention, specific activity (specific activity) minimum that discovery can prepare the final concentrated solution that comprises the IX thrombin is about the above highly purified IX NiaState of 150 (IU/mg).Should final concentrated solution put into as required behind the bottle freeze-dried, thereby as the experimental sample and the complete clinical sample of long-term safety.
Embodiment 7: the purifying of the IX thrombin in the cell culture medium
The Chinese hamster ovary celI of the IX factor will be cloned, after in serum free medium, cultivating, the 5L nutrient solution of Chinese hamster ovary celI will be removed, come purifying to prepare the IX factor according to removing the method that S/D (solvent/detergent) handles in the foregoing description 1～3 described method, the specific activity of this IX factor, purifying are doubly, output is as shown in table 7.
[table 7]
| Volume (ml) | Active (IU/ml) | Protein (mg/ml) | Gross activity (IU) | Gross protein (mg) | Specific activity (IU/mg) | Purifying doubly | Output (%) | |
| Cell culture fluid | 5000 | 2.1 | 1.25 | 10500 | 6250 | 1.68 | 1 | 100 |
| The resin anion(R.A) eluant | 500 | 17.3 | 2.75 | 8650 | 1375 | 6.29 | 3.7 | 82.4 |
| Affinity resin wash extract | 300 | 21.6 | 0.26 | 6480 | 78 | 83.08 | 49.5 | 61.7 |
| The non-adsorption liquid concentrated solution of resin cation (R.C.) | 100 | 54.0 | 0.38 | 5400 | 38 | 142.10 | 84.6 | 51.4 |
| Nanofiltration solution | 100 | 48.6 | 0.32 | 4860 | 32 | 151.88 | 90.4 | 46.3 |
| Dialysis concentrated solution (thick concentrated solution) | 100 | 43.1 | 0.28 | 4310 | 28 | 153.93 | 91.6 | 41.0 |
| Final concentrated solution | 100 | 40.6 | 0.26 | 4060 | 26 | 156.15 | 92.9 | 38.7 |
Embodiment 8: the purifying (inactivation) of virus and removal (removal) operation
In viral purification operation and nanofiltration operation, carry out many-sided virus and removed experiment (HIV; Human Immunodeficiency Virus, BHV; Bovine Herpes Virus, BVDV; Bovine Viral Diarrhea Virus, HAV; Hepatitis A Virus, EMCV; Encephalo Mycarditis Virus, PPV; Porcine Parvovirus), its test-results is shown in following table 8,9.Virus removal value in the table 8 is meant, in the titer that carries out stock virus, remove the LOG value of the data of titer, the virus of table 9 reduces value and is meant viral stock furcella (spike) behind target viral, the LOG value of the titer value after deducting operation finish from the data of carrying out furcella.
[table 8]
The virus that derives from the IX factor of blood plasma is removed operation
[table 9]
Folder comes from the virus of the IX factor of blood plasma and removes operation
The present invention relates to a kind of preparation method of highly purified people IX thrombin, wherein the material (taking from the cell culture medium of human plasma or reorganization) that comprises the IX thrombin is carried out the stage that ion exchange chromatography and heparin affinity chromatography make viral passivation or remove virus, can make the almost highly purified IX thrombin of inclusion-free, the safety of specific activity (specific activity) more than 150IU/mg.
More than, embodiments of the present invention are had been described in detail, but claim scope of the present invention is not only limited to above-mentioned scope, utilizes the various distortion of defined key concept in the claim scope of the present invention and improvement form to belong to claim scope of the present invention yet.
Claims (6)
1, a kind of preparation method of people IX thrombin is that raw material prepares people IX thrombin with the material that comprises the IX thrombin, and comprises the steps:
By carrying out the stage of anion-exchange chromatography collector IX thrombin more than 1 time;
By carrying out the stage that the heparin affinity chromatography carries out elution;
Collect the stage of non-adsorption liquid by carrying out cation-exchange chromatography;
Remove the stage of virus by carrying out nanofiltration.
2, the preparation method of people IX thrombin according to claim 1, wherein described by carrying out the stage that the heparin affinity chromatography carries out elution, the ionic strength (ionic strength) of employed level pad and the ionic strength that comprises the solution of the IX factor are below the 20mS/cm before being suitable for the solution comprise the IX factor, pH is 5.0～9.0, the ionic strength of the lavation buffer solution when washing is adsorbed in other protein of damping fluid is 10～20mS/cm, and the ionic strength of the elution damping fluid when elution comprises the solution of the IX factor is 20～50mS/cm.
3, the preparation method of people IX thrombin according to claim 1, the post functional group of wherein said cation-exchange chromatography is selected from carboxymethyl (carboxymethyl-, CM-), carboxyl (carboxy-, C-), sulfo group (sulfo-, S-), sulfonic acid methyl (sulfomethyl, SM-), sulfoethyl (sulfoethyl-, SE-), sulfopropyl (sulfopropyl-, SP-), phospho (phospho-, P-), post resin (resine) is selected from sepharose (Sepharose), dextran (Sephadex), agarose (agarose), Sephacel (Sephacel), polystyrene (Polystyrene), polyethylene (Polyacrylate), Mierocrystalline cellulose (Cellulose), Toyopearl.
4, the preparation method of people IX thrombin according to claim 1 wherein saidly collects the stage of non-adsorption liquid by carrying out cation-exchange chromatography, is to carry out under the condition of pH3.0～6.0.
5, the preparation method of people IX thrombin according to claim 1 wherein collects the stage of non-adsorption liquid described by carrying out cation-exchange chromatography, and the ionic strength that comprises the solution of the level pad and the IX factor is 10～50mS/cm.
6, the preparation method of people IX thrombin according to claim 1, the material that comprises the IX thrombin is the cell culture medium of the IX thrombin of human plasma or reorganization.
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| Application Number | Priority Date | Filing Date | Title |
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| KR10-2005-0098235 | 2005-10-18 | ||
| KR1020050098235A KR100667860B1 (en) | 2005-10-18 | 2005-10-18 | Method for manufacturing high purified factor ix product |
| PCT/KR2006/004237 WO2007046631A1 (en) | 2005-10-18 | 2006-10-18 | Method for manufacturing high purified factor ix |
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| CN101291951B CN101291951B (en) | 2012-07-04 |
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| KR (1) | KR100667860B1 (en) |
| CN (1) | CN101291951B (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104560925A (en) * | 2014-12-30 | 2015-04-29 | 山东泰邦生物制品有限公司 | Method for preparing human thrombin by activating human coagulation factor IX chromatography waste liquor |
| CN111378029A (en) * | 2018-12-29 | 2020-07-07 | 四川远大蜀阳药业有限责任公司 | Preparation method of high-stability and high-purity human coagulation factor IX |
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| AR083035A1 (en) | 2010-09-17 | 2013-01-30 | Baxter Int | STABILIZATION OF IMMUNOGLOBULINS THROUGH A WATERY FORMULATION WITH HISTIDINE AT WEAK ACID pH NEUTRAL, WATER COMPOSITION OF IMMUNOGLOBULIN |
| IL212911A0 (en) * | 2011-05-16 | 2011-07-31 | Omrix Biopharmaceuticals Ltd | Immunoglobulin reduced in thrombogenic contaminants and preparation thereof |
| KR20230136616A (en) * | 2014-02-04 | 2023-09-26 | 바이오젠 엠에이 인코포레이티드 | Use of cation-exchange chromatography in the flow-through mode to enrich post-translational modifications |
| CN115975997B (en) * | 2022-12-19 | 2023-11-17 | 成都蓉生药业有限责任公司 | Secondary ultrafiltration dialysate for purifying human coagulation factor IX and purification method |
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| US545718A (en) * | 1895-09-03 | Blast-furnace | ||
| DE3877529T2 (en) | 1987-10-23 | 1996-11-07 | Centre Regional De Transfusion | Production of highly purified human factor IX and other plasma protein concentrate and its therapeutic use. |
| DE4342132C1 (en) | 1993-12-10 | 1994-11-03 | Octapharma Ag | Process for the preparation of virus-inactivated vitamin K-dependent plasma components, and protein C and protein S-containing compositions by membrane chromatography |
| DE19504852C2 (en) | 1995-02-15 | 2003-01-23 | Octapharma Ag Lachen | Process for the purification of vitamin K-dependent coagulation factors by affinity chromatography |
| JPH09286797A (en) * | 1996-04-18 | 1997-11-04 | Green Cross Corp:The | Heparin cofactor ii and its purification |
| CN1297896A (en) * | 1999-11-29 | 2001-06-06 | 中国科学技术大学 | Method of extracting activated prothrombin X(FXa) effectively |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104560925A (en) * | 2014-12-30 | 2015-04-29 | 山东泰邦生物制品有限公司 | Method for preparing human thrombin by activating human coagulation factor IX chromatography waste liquor |
| CN104560925B (en) * | 2014-12-30 | 2017-11-03 | 山东泰邦生物制品有限公司 | A kind of method that activation prepares human thrombin in chromatography waste liquid from humanclottingfactorⅨ |
| CN111378029A (en) * | 2018-12-29 | 2020-07-07 | 四川远大蜀阳药业有限责任公司 | Preparation method of high-stability and high-purity human coagulation factor IX |
| CN111378029B (en) * | 2018-12-29 | 2023-05-05 | 四川远大蜀阳药业有限责任公司 | Preparation method of human coagulation factor IX |
Also Published As
| Publication number | Publication date |
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| WO2007046631A1 (en) | 2007-04-26 |
| CN101291951B (en) | 2012-07-04 |
| KR100667860B1 (en) | 2007-01-11 |
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